The proteasome plays a dominant role in the degradation of short-lived proteins and is a key mediator of biological regulation in all eukaryotes. It is the most complex proteolytic assembly known and is noteworthy for its self-compartmentalized structure. The proteasome is composed of a proteolytic core particle (CP) and a regulatory particle (RP). Substrates are thought to translocate from the RP into the lumen of the CP for degradation, traversing a narrow channel. The principal functions of the RP are proposed to be recognition of ubiquitin chains and ubiquitin-like proteins, unfolding and translocation of protein substrates, and gating of the channel of the CP. None of these functions is well-understood. In the previous funding period, we identified the 2.5-Megadalton proteasome holoenzyme of yeast, and determined the complete subunit composition of the 17-subunit RP, which includes six distinct ATPases. We also defined two functionally distinct subassemblies of the RP (the lid and the base), showed that the base links the RP to the CP, that the base has an ATP-dependent in vitro chaperone activity, implicated the base in gating of the channel into the proteolytic chamber of the CP, and identified the receptor for two ubiquitin-like proteins in the base. In the present proposal, we will extend these mechanistic studies. An important aspect of our strategy is that we have developed the capacity for rapid purification of proteasomes and its subcomplexes in milligram amounts. With the possibility of a closely integrated program of biochemical and genetic analysis, yeast provides an ideal model system for detailed analysis of this complex and remarkable enzyme. Specific Aims are as follows: 1. To purify and characterize subcomplexes from the RP. 2. To test whether the Rpn1 subunit of the base is a general receptor for ubiquitin-like proteins. 3. To identify the ubiquitin receptor of the proteasome. 4. To demonstrate the existence of a gated axial channel in the proteasome. 5. To characterize the chaperone-like activity of the proteasome.